Cell-Biology of Interferon Inducible GTPases

Cell-Biology of Interferon Inducible GTPases Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Sascha Martens aus Berlin Copy Team GmbH, Köln 2004

Berichterstatter: Prof. Dr. Jonathan C. Howard Prof. Dr. Thomas Langer Tag der mündlichen Prüfung: 9.7.2004

Für meine Familie und Anne

TABLE OF CONTENTS 1. INTRODUCTION... 1 1.1 RESISTANCE AND IMMUNITY... 1 1.2 CYTOKINES AND INTERFERONS... 2 1.3 THE INTERFERON SIGNAL TRANSDUCTION PATHWAY... 3 1.4 CELL-AUTONOMOUS IMMUNITY... 4 1.5 GTP BINDING PROTEINS AND MEMBRANE DYNAMICS... 6 1.6 DYNAMIN... 8 1.7 THE ANTIVIRAL MX PROTEINS... 9 1.8 OTHER INTERFERON INDUCIBLE GTPASES... 10 1.9 THE P47 GTPASES... 11 1.10 PHAGOCYTOSIS OF MICROBES... 15 1.11 THE AIM OF THIS STUDY... 16 2. MATERIAL AND METHODS... 18 2.1 REAGENTS AND CELLS... 18 2.1.1 Chemicals, Reagents and Accessories... 18 2.1.2 Equipment... 18 2.1.3 Materials... 18 2.1.4 Enzymes/Proteins... 19 2.1.5 Kits... 19 2.1.6 Vectors... 19 2.1.7 Cell lines... 19 2.1.8 Media... 20 2.1.9 Bacterial strains... 20 2.1.10 Serological reagents... 21 2.2 MOLECULAR BIOLOGY... 22 2.2.1 Agarose gel electrophoresis... 22 2.2.2 Generation of p47 GTPase expression constructs... 23 2.2.3 Cloning of PCR amplification products... 25

2.2.4 Purification of DNA fragments from agarose gels... 25 2.2.5 Ligation... 25 2.2.6 Preparation of competent cells... 26 2.2.7 Transformation of competent bacteria... 26 2.2.8 Plasmid isolation... 27 2.2.9 Determination of the concentration of DNA... 27 2.2.10 Site directed mutagenesis... 27 2.2.11 DNA Sequencing... 28 2.3 CELL BIOLOGY... 28 2.3.1 Transfections... 28 2.3.2 Bead uptake experiments... 29 2.3.3 Transferrin uptake experiments... 29 2.3.4 Indirect immunofluorescence... 29 2.3.5 Image acquisition and processing... 30 2.3.6 Triton X-114 partioning assay... 30 2.3.7 Membrane extraction experiments... 31 2.3.8 Preparation of artificial lipid vesicles... 32 2.3.9 Western Blotting... 32 2.3.10 Isolation of phagosomes... 32 2.3.11 Nucleotide agarose binding assay... 34 2.3.12 Preparation of infectious Salmonella typhimurium...34 2.3.13 Infection of B6m26 cells with Salmonella typhimurium... 34 2.3.14 In vitro passage of Toxoplasma gondii... 35 2.3.15 Preparation and culture of murine primary astrocytes... 35 2.3.16 In vitro infection experiments and inoculation of primary astrocytes with Toxoplasma gondii... 36

3. RESULTS... 27 3.1 THE P47 GTPASES ARE INDUCED BY IFN-γ IN L929 AND TIB-75 CELLS.... 37 3.2 THE P47 GTPASES SHOW DIFFERENT LEVELS OF MEMBRANE ASSOCIATION.... 37 3.3 LRG-47 BINDS GDP AND LOCALIZES TO THE GOLGI APPARATUS AND THE ER... 38 3.4 LRG-47 DOES NOT ASSOCIATE WITH THE ENDOSOMAL OR LYSOSOMAL COMPARTMENT... 43 3.5 LRG-47 IS RECRUITED TO THE PLASMA MEMBRANE UPON PHAGOCYTOSIS AND REMAINS ASSOCIATED WITH MATURING PHAGOSOMES.... 45 3.6 MEMBRANE BINDING OF LRG-47 IS IFN-γ AND NUCLEOTIDE INDEPENDENT... 48 3.7 LRG-47 IS TARGETED TO THE GOLGI APPARATUS AND PLASMA MEMBRANE BY DIFFERENT DOMAINS.. 51 3.8 THE αk REGION OF LRG-47 IS SUFFICIENT FOR GOLGI TARGETING... 52 3.9 THE GOLGI TARGETING ACTIVITY OF THE LRG-47 αk REGION REQUIRES AN AMPHIPATHIC HELIX.... 54 3.10 THE αk REGIONS OF GTPI AND IGTP ALSO SHOW MEMBRANE TARGETING ACTIVITY... 56 3.11 THE αk PEPTIDES OF GTPI AND IGTP SHOW HOMOLOGY TO TWO DIFFERENT REGIONS OF PHOSPHOLIPASE C.... 57 3.12 THE αi, J REGION LRG-47 SHOWS HOMOLOGY TO HUMAN PHOSPHOLIPASE C LIKE PROTEIN... 59 3.13 IIGP1 IS ASSOCIATED WITH THE ER.... 60 3.14 IIGP1 FORMS NUCLEOTIDE AND C-TERMINAL DOMAIN DEPENDENT AGGREGATES AFTER TRANSFECTION... 60 3.15 IIGP1 IS N-TERMINALLY MYRISTOYLATED.... 63 3.16 IIGP1 IS TARGETED TO ENDOMEMBRANES BY THE N-TERMINAL DOMAIN AND TO THE PLASMA MEMBRANE BY THE G-DOMAIN.... 65 3.17 THE P47 GTPASES BIND TO MEMBRANE WITH DIFFERENT STRENGTH... 68 3.18 THE MYRISTOYL-GROUP OF IIGP1 DOES NOT CONTRIBUTE TO THE STRENGTH OF MEMBRANE ASSOCIATION... 68 3.19 LOCALIZATION OF TAGGED GMS GTPASES IN L929 CELLS... 69 3.20 EXPRESSION OF TGTP IN CELLS... 71 3.21 EXPRESSION OF IIGP1-HIS IN CELLS... 74 3.22 IIGP1 ACCUMULATES AT TOXOPLASMA GONDII CONTAINING PARASITOPHOROUS VACUOLES... 76 3.23 IGTP LOCALIZES TO TOXOPLASMA GONDII CONTAINING PARASITOPHOROUS VACUOLES.... 78

4. DISCUSSION... 81 4.1 THE INTERFERON INDUCIBLE P47 GTPASES ARE REMARKABLY DIVERSE REGARDING THEIR MEMBRANE ASSOCIATION PROPERTIES.... 81 4.2 LRG-47 BINDS GDP AND IS A GOLGI ASSOCIATED PROTEIN... 82 4.3 LRG-47 IS RECRUITED TO PHAGOSOMAL ENVIRONMENT AND PLASMA MEMBRANE EARLY UPON PHAGOCYTOSIS... 84 4.4 DOMAINS RESPONSIBLE FOR THE DYNAMIC INTRACELLULAR BEHAVIOUR OF LRG-47... 86 4.5 LRG-47 IS TARGETED TO GOLGI BY A C-TERMINAL AMPHIPATHIC HELIX.... 87 4.6 THE αk REGIONS OF ALL MOUSE GMS GTPASES MEDIATE MEMBRANE ASSOCIATION.... 89 4.7 THE ER PROTEIN IIGP1 SHOWS STRIKING DIFFERENCES TO LRG-47... 91 4.8 TRANSFECTION AND EXPRESSION OF P47 GTPASES IN CELLS... 95 4.9 THE DYNAMICS OF P47 GTPASES IN INFECTED CELLS... 97 4.10 MODELS FOR P47 GTPASE FUNCTION... 100 5. REFERENCES... 105 6. SUMMARY... 123 7. ZUSAMMENFASSUNG... 124 8. DANKSAGUNGEN... 126 9. ERKLÄRUNG... 127 10. LEBENSLAUF... 128

ABBREVATIONS 2'-5'-OAS 2'-5'-oligoadenylate synthetase ADAR adenosine deaminases that act on double-stranded RNA APC antigen presenting cell APOBEC3G apolipoprotein B mrna-editing, enzyme-catalytic, polypeptide-like 3G APS ammoniumpersulfate ATP adenosine triphosphate BAC bacterial artificial chromosome bp base pair BSA bovine serum albumine CI-MP6R cation-independent mannose 6-phosphate receptor C-terminal carboxy terminal DMEM Dulbecco Modified Eagles Medium DMSO dimethylsulfoxid DNA desoxyribonucleicacid E. coli Escherichia coli EDTA ethylendiamintetraacetic acid ER endoplasmatic reticulum EtBr ethidium bromide EtOH ethanol FCS foetal calf serum GAP GTPase activating protein GBP guanylate binding protein GEF guanine nucleotide exchange factor GDI guanine nucleotide dissociation inhibitor GDP guanosine diphosphate GMP guanosine monophosphate GTP guanosine triphosphate IB immuno blot IDO indoleamine 2,3-dioxygenase IF immunofluorescence IFN-γ Interferon-γ IFN-α/β Interferon-α/β IFNGR IFN-γ receptor IFNAR IFN-α receptor inos inducible nitric oxide synthetase NRAMP1 natural resistance associated membrane protein 1 IRF-1 Interferon regulatory factor 1 IP 3 inositol-l,4,5-trisphosphate ISG20 interferon stimulated gene 20 JAK janus kinase kb kilobase kda kilodalton LAMP-1 lysosome associated membrane protein 1 LPS lipopolysaccharide M molar MEF mouse embryonic fibroblats MOPS 3-[ Morpholino]propansulfonsäure N-terminal amino-terminal OD optical density ON over night ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction PFA paraformaldehyde PH pleckstrin homology domain PLC phospholipase C phox phagosome oxidase PIK phosphatidyl inositol kinase PIP phosphatidyl inositol phosphate

PKR protein kinase R PML promyelocytic leukaemia PtdInsP phosphatidyl inositol phosphates RNAse ribonuclease rpm rounds per minute RT room temperature RNAi RNA interference S. typhimurium Salmonella typhimurium SDS sodium dodecylsulfate SDS-PAGE SDS polyacrylamide gel electrophoresis SSH suppressive subtractive hybridization STAT signal transducer and activator of transcription TEMED N,N,N,N Tetramethyldiamine T. gondii Toxoplasma gondii TGN trans-golgi network Tris/HCl Tris[hydroxymethyl]aminoethane TRITC tetramethylrhodamine isothiozyanate TNF-α tumor necrosis factor α TRIM5α tripartite motif 5α U unit UV ultraviolet VOL volume WT wild type ZAP Zinc-finger Antiviral Protein Units ' minute '' second Ø diameter % percent % solution grams in 100ml solution C degree Celsius aa amino acids bp base pair Ci Curie (1 Ci = 3.7x107 cpm) cpm counts per minute g G force g gram h hour kb kilo bases l Litre LD lethal doses m meter M molar m2 square meter nt nucleotide OD optical density rpm rounds per minute t time U unit v/v weight pro volume c centi, 10-2 m milli, 10-3 µ micro, 10-6 n nano, 10-9 p pico, 10-12

Introduction 1. Introduction 1.1 Resistance and Immunity Resistance to infectious agents is a common property of all living things in order to maintain their structural integrity. Pathogens try to invade the host, disseminate and establish infection to exploit the organism for its own advantage. The host in contrast attempts to intervene in the pathogens actions at every possible step vulnerable to attack. Hundreds of million years of reciprocal adaptation have led to a complex many layered and interconnected system of interactions between host and pathogen. These interactions often led to a relationship in which action and counteraction are well balanced and infections become only visible, or even possible, upon impairment of host resistance. A remarkable proportion of the genome of both players is devoted to these interactions (1-5). Central to prevention and containment of infectious agents is their recognition. This is achieved by receptors sensing molecules invariably associated with pathogens and/or their actions (6, 7). The list of receptors recognizing pathogen associated patterns (PAMPs) is long and ever growing including the Toll-like receptors (6), Nod receptors (8, 9), lectins (10) and double-stranded RNA recognizing molecules (11, 12). These receptors are found throughout life including plants and mammals. The activation of these receptors elicits the induction or activation of defence programs counteracting infections in a diverse manner (13, 14). The expression of pattern recognition receptors (PRRs) is in principle not restricted to special cell types but is particularly eminent on phagocytic cells such as macrophages (10). In vertebrates, the adaptive immune system builds a second, highly sophisticated resistance system. In contrast to the innate immune system which utilizes the aforementioned receptors, adaptive immunity is based on receptors which are clonally rearranged in the soma. In their entirety they are potentially able to recognize virtually every chemical component in the appropriate context. B-cells carry special receptors (BCR) which induce, upon their activation, clonal expansion and production of antibodies targeting extracellular pathogens. T-cells, and in particular CD8 positive T- cells, recognize intracellular pathogens by means of their T-cell receptor (TCR). Upon activation T-cells kill the infected cell or secrete factors inducing the infected cell to neutralize the pathogen by itself in a non-cytolytic manner (15, 16). 1

Introduction Upon the first encounter of a pathogen it takes several days for the adaptive immune system to mount an effective and robust immune response. The innate immune system in contrast is able to respond to invading microbes within minutes and most infections are therefore contained within a short time. However in many cases the response to infections involves cells of both systems and is orchestrated by small soluble molecules called cytokines. 1.2 Cytokines and Interferons Cytokines are small soluble proteins which belong to four, structurally distinct, classes namely the haematopoins, the chemokines, members of the TNF (tumor necrosis factor) family and the interferons (IFN). Although the secretion is not limited to them, major producers of cytokines are professional immune cells. Numerous cellular responses such as proliferation, chemotaxis but also angiogenesis and embryogenesis are regulated by cytokines (17). Some cytokines, and in particular interferons, have the remarkable ability to elicit extraordinarily complex cellular responses virtually changing the whole physiology of the responding cell (18-20). Interferons are subdivided into three types. Type I interferons include IFN-α with 10 members and IFN-β (21), IFN-ω (22), IFN-κ (23), IFN-δ (24) with a single member each. Type II interferon has only one member, IFN-γ and finally the recently discovered IFN-λ (25, 26) having three members. Type I interferons are secreted by many cell-types including fibroblasts and dendritic cells (DCs) (27, 28). The main stimulus of type I interferon production is virus infection and consequently its main function is the induction of an antiviral state in the responding cell (12). However the action of type I interferons is not restricted to viral purging (19). IFN-γ is secreted by activated T-cells (29, 30), NK-cells (31) and macrophages (32). The cellular response to IFN-γ is enormously complex (18-20) regulating a considerable percentage of the genome (1). Thus it is not surprising that the actions of IFN-γ are diverse and play an important role in innate and adaptive immunity. 2

Introduction 1.3 The interferon signal transduction pathway Type I and type II interferons are recognized by different receptors (Figure I1). Biologically active IFN-γ is a homodimer and binds to the heterodimeric IFN-γ receptor (IFNGR1 and IFNGR2) expressed on all nucleated cells. Binding of IFN-γ to its receptor induces receptor dimerization and thereby a cascade of intracellular tyrosine phosphorylation events involving JAK1 and JAK2 ultimately leading to the activation and nuclear translocation of a STAT1 transcription factor homodimer called GAF (gamma activated factor) (33). In the nucleus GAF binds to gamma-interferon activated sequence (GAS) elements found at the promoter of many interferon responsive genes leading to their activation. Figure I1: Simplified scheme of the type I and Type II signal transduction pathways taken from (34). Binding of type I interferon (IFN-α) to its receptor induces dimerization of the IFNAR1 and IFNAR2 receptor subunits, activation of the associated JAK1 and TYK2 proteins subsequent phosphorylation of STAT1 and STAT2 and thereby their dimerization. The STAT1/STAT2 dimer translocates into the nucleus and forms a trimeric complex with IRF9 which binds to ISRE elements at the promoter of IFN stimulated genes and promotes their transcription. Binding of type II IFN (IFN-γ) to its receptor induces its dimerization, activation of JAK1 and JAK2 which is followed by phosphorylation of STAT1 and its homodimerization. The STAT1 homodimer binds to GAS elements at the promoter of IFN-γ stimulated genes after translocation into the nucleus. Among the primary response genes are further transcription factors such as IRF-1 leading to a second wave of gene expression (18). The signal transduction pathway of type I IFN is very similar to that of IFN-γ. Binding of IFN-α or IFN-β to its heterodimeric receptor (IFNAR1 and IFNAR2) induces receptor dimerization, activation of JAK1 and TYK2 and subsequent phosphorylation of 3

Introduction STAT1 and STAT2. The STAT1/STAT2 heterodimer associates with IRF-9 to form a heterotrimeric complex called ISGF3. ISGF3 binds to ISRE (interferon stimulated response elements) near the promoter of IFN activated genes. Type I and type II IFNs activate distinct but overlapping sets of genes (19, 35-37). Experiments with mice carrying targeted deletions of components of the IFN-γ and IFNα/β signal transduction pathways respectively have shown that IFN-γ is essential for resistance against bacterial and protozoan pathogens having a rather mild effect on certain viral infections. The opposite effect was observed for IFN-α/β which is central to combat viral infections but has a comparably moderate effect on bacterial and protozoan infections (38, 39). Due to its complexity the function of IFN regulated genes is hard to assess not least because the function of many inducible genes is unknown. However, in general IFN inducible genes play a role in one or more of the following cellular programs (18, 19). This includes the regulation of immune cell function, regulation of proliferation, enhancement and modulation of antigen-presentation and direct anti-microbial effects. Many of the direct anti-microbial effects are mediated in a cell-autonomous manner. 1.4 Cell-autonomous immunity It is becoming increasingly clear that cell-autonomous immunity plays a major role in resistance to intracellular pathogens of all classes. Many of the molecular players of intracellular resistance are inducible by interferons. The main focus has been on factors restricting viral growth (12) but it is now appreciated that also bacteria and protozoa are restricted by cell-autonomous resistance mechanisms (40). By definition cellautonomous immunity is mediated by a cell for itself without the requirement for other specialized cell types. However, the induction of cell-autonomous resistance in a particular cell might be dependent on specialized cells as exemplified by the production of IFN-γ. Indeed it turned out that production of IFN-γ is used by activated T-cells to induce cell-autonomous immunity in the target cell and thereby to clear viral infections (16, 15). The list of proteins implicated in cell-autonomous immunity is long and still growing (41). The following table includes an incomplete overview of factors known to play an important role in cell-autonomous immunity (Table 1). Many of the listed proteins have in addition functions different from direct anti-microbial effects. PKR for example has 4

Introduction been shown to be central for signal transduction in a variety of cellular pathways (11) (42) or to mediate general functions in membrane traffic like Rab5a (43, 44). However many of the included proteins have no known function besides pathogen resistance. Table 1 NAME SPECIES TARGET LOCATION REFERENCE Mx vertebrates Bunyaviridae, nucleus, cytoplasm (45-48) Orthomyxoviridae (smooth ER) 2-5 OAS, RNaseL mouse, human Picornaviridae nucleus, cytoplasm (12, 49) PKR mouse, human EMCV, Vaccinia cytoplasm (50-53) Virus, VSV ADAR1 human Hepatitis delta virus nucleus, cytoplasm (54, 55) ISG20 human VSV, Influenza virus, nucleus (56, 57) EMCV p65 GTPases vertebrates VSV, EMCV cytoplasm (58-60) PML mouse, human VSV, influenza virus, nucleus (61-63) human foamy virus, HSV1 ZAP rat Murine leukaemia ND (64) virus (MLV) CEM15/APOBEC3G mammals Hepatitis B Virus and cytoplasm (65-70) retroviruses including HIV, SIV, MLV, EIAV TRIM5α primates HIV, SIV cytoplasmic bodies (41) RNAi machinery eukaryotes dsrna viruses cytoplasm (71-75) including HIV-1, HCV, Poliovirus, Hepatitis delta virus FV1, Ref1, Lv1 rodents, primates retroviruses including Golgi, ER (76-79), FV1, MLV, HIV-1, HIV-2, EIAV IDO mouse, human viruses, bacteria and cytosol (80-83) protozoa including cytomegalovirus, Clamydia, Toxoplasma gondii inos vertebrates over 80 pathogens of cytoplasm (84-87) all classes phox complex vertebrates pathogens of viral, phagosomal membrane (88) bacterial and protozoan origin NRAMP1 mouse, human Salmonella, phagosomal membrane (89-91) Leishmania, Mycobacterium p47 GTPases mouse bacterial and protozoan ER, Golgi, cytosol (40, 92-96), this study pathogens Rab5a eukaryotes Listeria early endosomes (43) 5

Introduction The diversity of factors included in the table (Table 1) reflects the diverse and complex intracellular behaviours of the respective pathogens. It is conspicuous that many of the proteins targeting bacterial and protozoan pathogens are membrane-bound. Intracellular membranes are central for the survival of many pathogens and therefore it is not surprising that pathogen and host attempt to take over control of cellular membrane dynamics (97). In eukaryotic cells many of the essential steps involving membrane dynamics are mediated by GTP binding proteins (10, 44, 98). 1.5 GTP binding proteins and membrane dynamics GTP binding proteins are central to a plethora of cellular functions including protein biosynthesis, transport across the nuclear envelope, signal transduction and membrane traffic. Despite such diverse functions the underlying mechanism is the same for all GTPases involving a conformational change upon binding of GTP and subsequent hydrolysis of the nucleotide to GDP and/or GMP (99-102). The energetically favourable reaction is able to create order or force. Guanine nucleotide binding is essential for the function of GTPases and involves 5 motifs called G1-G5 among which G1, G3 and G4 are universally conserved. The consensus sequence of the G1 motif is GX 4 GKS contacting the α-, β- and γ-phosphate of the bound nucleotide. The G3 motif makes contact to the γ-phosphate and the G4 motif confers specificity by contacting the base of the guanine nucleotide (99-101). For the switch GTPases the GTP bound form is considered active and interacts with other molecules termed GTPase effectors. This interaction is responsible for the downstream effects of the GTP bound GTPase. Inactivation is achieved by hydrolysis of GTP and dissociation of the γ phosphate. Many GTPases have a low intrinsic activity and inactivation is mediated by the action of GTPase activating proteins (GAPs) which accelerate hydrolysis. The resulting GDP bound form is considered inactive. In many cases GTPases become activated upon interaction with guanine nucleotide exchange factors (GEFs). GEFs mediate dissociation of GDP from the GTPase. Due to the 3 fold higher concentration of GTP within the cell (103) GTPases become thereby activated despite often similar affinities for GTP and GDP (100). Some GTPases like the Rab and Rho are additionally regulated by guanine nucleotide dissociation inhibitors (GDIs) 6

Introduction which bind to the GDP bound form and prevent nucleotide dissociation (104). A simplified cartoon summarizing the GTPase cycle is shown in Figure I2. Figure I2: Simplified scheme of the GTPase cycle. Activation of the GDP bound GTPase by a trigger is achieved by exchange of GDP for GTP. This exchange is often mediated by the activation of exchange factors (GEF). The GTP bound GTPase interacts with molecules termed effectors mediating its downstream effects. GTP hydrolysis and dissociation of the γ-phosphate leads to the inactivation of the GTPase. The often low intrinsic hydrolytic activity can be accelerated by activating proteins (GAP). Some GDP bound GTPases are kept inactive by the action of guanine nucleotide dissociation inhibitors (GDI) (99-101). Due to their ability to be regulated in multiple ways and to give complex reactions a direction, GTPases are central regulators and mediators of membrane traffic and membrane association is essential for their function. Arf GTPases for example regulate COP mediated vesicular budding (105). The inactive GDP bound form is cytosolic but upon nucleotide exchange from GDP to GTP Arf exposes an N-terminal myristoyl-group and an amphipathic helix hidden within the molecule anchoring the protein in the lipid bilayer (106, 107). At the membrane Arf interacts with other molecules to initiate vesicular budding (105). Rab GTPases in contrast are isoprenylated at their C-terminus and are kept soluble in the cytosol by the interaction with RabGDI burying the large C-terminal isoprenyl-group in a hydrophobic pocket (44, 105). Activation of Rab by nucleotide exchange from GDP to GTP leads to dissociation of Rab from RabGDI and subsequent membrane association. 7

Introduction At the membrane Rab GTPases interact with various effectors thereby determining specificity of vesicular transport and organelle identity (44). Rho GTPases are yet another family of GTP binding proteins belonging, like Arf and Rab, to the p21 Ras superfamily of GTPases. Rho GTPases such as RhoA, CDC42 and Rac1 regulate actin dynamics (108-110) and function in phagosome formation (111-113) and maturation by recruiting the phagosome oxidase complex to the phagosomal membrane (114). In addition they have various other cellular functions (108). Activation of Rho GTPases results in membrane association which is, as in the case of Rab, mediated by a C-terminal isoprenyl-group (115). The GDP bound form of Rho GTPases is found in the cytosol in a complex with RhoGDI (116). Upon exchange of GDP for GTP Rho GTPases bind to intracellular membranes collectively called endomembranes and the plasma membrane (117, 118). In all cases nucleotide exchange and membrane association are strictly coupled. 1.6 Dynamin Dynamins are GTPases which mediate scission of vesicles budding from the donor membrane. Overexpression of dominant negative mutants of dynamin interferes with the formation of clathrin-coated vesicles, budding from caveolae and phagosome formation. Dynamins differ in several key aspects from the p21 Ras superfamily of GTPases (98, 102, 119). Dynamins are with a molecular weight of about 100 kda much larger than Arf, Rab and Rho GTPases which are 20-30 kda in size. Since their discovery it is under debate whether dynamins function by generating force or are molecular switches analogous to the members of the Ras superfamily of GTPases, or both (98, 102, 120-122). The N-terminal nucleotide binding domain of dynamin is followed by the middle domain implicated in self-assembly, a pleckstrin homology domain (PH domain) involved in membrane targeting, a GTPase effector domain accelerating GTP hydrolysis and a C-terminal proline-rich domain interacting with other proteins (98). Although many proteins have been shown to interact with dynamin no proteins, apart from dynamin itself, could be identified so far interacting with dynamin in a nucleotide dependent manner (98, 102). Compared to Ras-like GTPases dynamins bind guanine nucleotides with a rather low affinity in the µm range and have high turn over rates of hydrolysis. Dynamin self associates in a GTP dependent manner which increases the specific activity of the dynamin subunits (123). Thus nucleotide hydrolysis of dynamin 8

Introduction is cooperative. Dynamin associates with negatively charged liposomes in vitro and this association accelerates hydrolysis about 100 fold (123, 102). In the presence of GTP-analogues dynamin tubulates lipids in vitro by forming ring-like structures around liposomes (124). Upon hydrolysis the diameter of the tubulated liposomes decreases (122). In vivo dynamin was observed to form spiral-like structures around the neck of budding vesicles (124). The presence of lipids massively accelerates GTP hydrolysis and enhances nucleotide dependent oligomerization and self-assembly (102, 123). Membrane binding is essential for the function of dynamin itself and probably also for the other members of the dynamin superfamily of which the mammalian dynamin 1, 2 and 3 are the prototypes (98). The PH domain of dynamin is involved in targeting of dynamin to membranes mediated by its low affinity for the lipid head group inositol 1,4,5-triphosphate. Mutations in the PH domain of dynamin have a dominant negative effect on endocytosis (125-127). However other members of the dynamin superfamily lack a PH domain but are still capable of lipid binding as for example the Mx proteins (128, 45). 1.7 The antiviral Mx proteins Mouse Mx was the first member of the dynamin superfamily discovered and was initially identified as a dominant locus in A2G mice conferring resistance to infections by orthomyxoviridea (129). Mapping and subsequent cloning of the gene led to the identification of mouse Mx1 (48, 130) and the human homologue MxA (131). Surprisingly, in contrast to most out bred mice, most laboratory mouse strains do not carry a functional allele of Mx (132, 133). Mx proteins exhibit nucleotide dependent oligomerization and cooperative hydrolysis (47, 134, 135). A direct interaction of Mx with viral particles has been shown and is proposed to be important for its antiviral activity (47). However a mutant Mx protein, defective in GTP hydrolysis and oligomerization still shows antiviral activity questioning the role of self assembly for its antiviral function (136). Recently human MxA has been reported to localize to the smooth ER and to tubulate lipids in a nucleotide dependent manner (45). The meaning of membrane deformation by MxA for its biological function however is unclear recalling that dynamin has been first isolated as a protein assembling around microtubules (137) but there is still no in vivo function described giving this finding significance. A hallmark of the Mx proteins 9

Introduction is their exclusive inducibility by type I IFN (138) and low or absent level in resting cells. 1.8 Other Interferon inducible GTPases The induction of high molecular weight GTPases by IFNs turns out to be an important component of their biological function. There are at least three more families of GTP binding proteins which are massively induced by IFN and whose functions are dedicated to host resistance. The p65 family of GTPases has 5 members (GBP1-5) in mouse and human (139). Homologues are found in all vertebrates analyzed so far (60). The p65 GTPases are abundantly induced by type I and type II IFN from low resting levels (60). When expressed in Hela cells human GBP1 (hgbp1) shows a cell-autonomous antiviral effect against VSV and EMCV (58). Besides its antiviral activity members of the p65 family have been implicated in the regulation of cell proliferation (140-142). The crystal structure of hgbp1 reveals a three domain protein having an N-terminal GTP binding domain, followed by a helical middle domain and a C-terminal GTPase effector domain (143). Some family members are isoprenylated mediated by a C-terminal CAAX motif (C, cysteine; A, large hydrophobic residues; X, any residue) and this isoprenylation appears to be responsible for the localization in enigmatic cytoplasmic dots (59) (H. Kashkar, unpublished results). The structure of hgbp1 has attracted much attention because it is generally believed that its overall structural organization is shared with dynamin (143). Recently a family of gigantic GTP binding proteins with a molecular weight of 280 kda has been published (144). This very large inducible GTPases (VLIGs) are massively induced by type I and type II IFNs, at least in the mouse. No anti-microbial effect for the VLIGs has been shown so far but VLIG-1, the prototype of the VLIG family, shows highest homology to GTPases mediating cell-autonomous resistance within the GTPase superfamily (144). This suggests, in addition to their IFN inducibility, a role in intracellular defence. The third family of IFN inducible GTP binding proteins are the p47 GTPases. 10

Introduction 1.9 The p47 GTPases There is now compelling evidence that the p47 GTPases are an essential component of the immune response against intracellular pathogens in the mouse (40). So far 6 p47 GTPases have been published, namely TGTP (145), IRG-47 (146), IIGP1 (60), IGTP (147), GTPI (60) and LRG-47 (148) but the total number of p47 genes in the Mus musculus domesticus genome is 23 of which 4 are pseudogenes by one or another criterion (Julia Hunn, Cemali Bekpen and Jonathan C. Howard, personal communication). Figure I3 shows a phylogeny of the p47 GTPases of Mus musculus domesticus. TGTP subfamily IIGP subfamily GMS subfamily Figure I3: Phylogenetic Tree based on the protein sequence of the GTPase domain of the mouse p47 GTPases (courtesy of Cemali Bekpen) showing 22 out of the 23 members. The neighbour joining tree is based on the distance method using the MEGA2 program. The phylogeny reveals a complex protein family which can be subdivided into several subgroups namely the TGTP, IIGP and GMS subfamily. In addition, the phylogeny 11